Reduced glomerular filtration rate (GFR) in chronic kidney disease (CKD) results in an accumulation of a large number of metabolites, a subset of which likely mediate risk of end-stage renal disease (ESRD). In this proposal, we will discover and validate blood metabolites whose levels predict ESRD risk beyond that marked by low GFR in 5 CKD studies. Highest emphasis will be placed on metabolomic abnormalities which are promising therapeutics targets and/or outcomes for CKD clinical trials. Study aim summary: Aim 1: To discover metabolic risk factors for CKD progression through global metabolomic profiling. Unbiased global metabolomic profiling now allows reliable quantification of ~1,000 named metabolites from a large compound library. We will apply these methods to samples from AASK (n=804, 296 incident ESRD), MDRD Study (n=697, 546 incident ESRD) and CRIC (case-cohort design, n=500 with 250 incident ESRD cases). We hypothesize that a subset of metabolites with altered levels in CKD will remain risk factors for ESRD after rigorously adjusting for baseline GFR. These metabolites will also hold great promise for risk stratification and as therapeutic targets (e.g., to diet and microbiome manipulation). Aim 2: To validate findings from Aim 1 in an independent sample and extend analyses to other CKD etiologies (polycystic kidney disease, PKD; kidney transplantation, KT). Validation will focus on four methodological aspects: 1) longitudinal stability in follow-up samples (n =50 per study); 2) risk associations of total vs. free metabolite levels, with the latter determined by removal of protein- bound metabolites; 3) external replication in additional samples in a general CKD cohort, CRIC (N=400); and 4) replication in 2 CKD cohorts with distinct etiologies (HALT-PKD and FAVORIT (KT); N=400 each). Aim 3: To characterize the effect of randomized dietary interventions on the metabolome in CKD Using samples at baseline and after randomization we will test the hypothesis that randomized interventions in MDRD Study (low protein diets and essential keto-acids) and FAVORIT (folate and B-vitamin) dramatically alter the metabolome; and that metabolite alterations will relate to subsequent outcomes and may explain subgroups where the intervention was effective, although the trials were negative overall. Aim 4: To develop, evaluate, and apply clinical assays for validated metabolites Targeted assays provide the absolute quantification and greater precision needed for clinical application. We will develop clinical laboratory tests to CAP (College of American Pathologists) standards for the top hits from Aim 2, re-measure a subsample from each cohort and test in a new clinical cohort (n=800). The proposal benefits from our extensive history of innovation and collaboration in CKD-Biocon as well as CKD clinical guideline development, CKD Epidemiology Collaboration, CKD Prognosis Consortium, CKD genetics, and FDA workshops and can result in great advances in the understanding and treatment of CKD progression.